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1.
Viruses ; 13(11)2021 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-34834977

RESUMO

Yersinia enterocolitica is a food-borne Gram-negative pathogen responsible for several gastrointestinal disorders. Host-specific lytic bacteriophages have been increasingly used recently as an alternative or complementary treatment to combat bacterial infections, especially when antibiotics fail. Here, we describe the proteogenomic characterization and host receptor identification of the siphovirus vB_YenS_ϕR2-01 (in short, ϕR2-01) that infects strains of several Yersinia enterocolitica serotypes. The ϕR2-01 genome contains 154 predicted genes, 117 of which encode products that are homologous to those of Escherichia bacteriophage T5. The ϕR2-01 and T5 genomes are largely syntenic, with the major differences residing in areas encoding hypothetical ϕR2-01 proteins. Label-free mass-spectrometry-based proteomics confirmed the expression of 90 of the ϕR2-01 genes, with 88 of these being either phage particle structural or phage-particle-associated proteins. In vitro transposon-based host mutagenesis and ϕR2-01 adsorption experiments identified the outer membrane vitamin B12 receptor BtuB as the host receptor. This study provides a proteogenomic characterization of a T5-type bacteriophage and identifies specific Y. enterocolitica strains sensitive to infection with possible future applications of ϕR2-01 as a food biocontrol or phage therapy agent.

2.
Viruses ; 13(7)2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34372590

RESUMO

Bacteriophages vB_YpeM_fEV-1 (fEV-1) and vB_YpeM_fD1 (fD1) were isolated from incoming sewage water samples in Turku, Finland, using Yersinia pestis strains EV76 and KIM D27 as enrichment hosts, respectively. Genomic analysis and transmission electron microscopy established that fEV-1 is a novel type of dwarf myovirus, while fD1 is a T4-like myovirus. The genome sizes are 38 and 167 kb, respectively. To date, the morphology and genome sequences of some dwarf myoviruses have been described; however, a proteome characterization such as the one presented here, has currently been lacking for this group of viruses. Notably, fEV-1 is the first dwarf myovirus described for Y. pestis. The host range of fEV-1 was restricted strictly to Y. pestis strains, while that of fD1 also included other members of Enterobacterales such as Escherichia coli and Yersinia pseudotuberculosis. In this study, we present the life cycles, genomes, and proteomes of two Yersinia myoviruses, fEV-1 and fD1.


Assuntos
Bacteriófagos/genética , Bacteriófagos/fisiologia , Genoma Viral , Proteoma , Yersinia pestis/virologia , Bacteriófagos/ultraestrutura , Finlândia , Especificidade de Hospedeiro , Microscopia Eletrônica de Transmissão , Esgotos , Yersinia pestis/classificação
3.
Mol Cancer Ther ; 20(10): 1996-2007, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34315766

RESUMO

Polysialic acid, an abundant cell surface component of the developing nervous system, which declines rapidly postnatally to virtual absence in the majority of adult tissues, is highly expressed in some malignant tumors including neuroblastoma. We found that the binding of a noncatalytic endosialidase to polysialic acid causes internalization of the complex from the surface of neuroblastoma kSK-N-SH cells, a subline of SK-N-SH, and leads to a complete relocalization of polysialic acid to the intracellular compartment. The binding and uptake of the endosialidase is polysialic acid-dependent as it is inhibited by free excess ligand or removal of polysialic acid by active endosialidase, and does not happen if catalytic endosialidase is used in place of inactive endosialidase. A fusion protein composed of the noncatalytic endosialidase and the cytotoxic portion of diphtheria toxin was prepared to investigate whether the cellular uptake observed could be used for the specific elimination of polysialic acid-containing cells. The conjugate toxin was found to be toxic to polysialic acid-positive kSK-N-SH with an IC50 of 1.0 nmol/L. Replacing the noncatalytic endosialidase with active endosialidase decreased the activity to the level of nonconjugated toxin. Normal nonmalignant cells were selectively resistant to the toxin conjugate. The results demonstrate that noncatalytic endosialidase induces a quantitative removal and cellular uptake of polysialic acid from the cell surface which, by conjugation with diphtheria toxin fragment, can be exploited for the selective elimination of polysialic acid-containing tumor cells.

4.
Viruses ; 13(5)2021 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-33923360

RESUMO

Bacteriophage vB_EcoM_fHy-Eco03 (fHy-Eco03 for short) was isolated from a sewage sample based on its ability to infect an Escherichia coli clinical blood culture isolate. Altogether, 32 genes encoding hypothetical proteins of unknown function (HPUFs) were identified from the genomic sequence of fHy-Eco03. The HPUFs were screened for toxic properties (toxHPUFs) with a novel, Next Generation Sequencing (NGS)-based approach. This approach identifies toxHPUF-encoding genes through comparison of gene-specific read coverages in DNA from pooled ligation mixtures before electroporation and pooled transformants after electroporation. The performance and reliability of the NGS screening assay was compared with a plating efficiency-based method, and both methods identified the fHy-Eco03 gene g05 product as toxic. While the outcomes of the two screenings were highly similar, the NGS screening assay outperformed the plating efficiency assay in both reliability and efficiency. The NGS screening assay can be used as a high throughput method in the search for new phage-inspired antimicrobial molecules.


Assuntos
Bacteriófagos/genética , Toxinas Biológicas/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Bacteriófagos/isolamento & purificação , Bacteriófagos/ultraestrutura , Biologia Computacional/métodos , Genoma Viral , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Especificidade de Hospedeiro , Humanos , Modelos Moleculares , Conformação Proteica , Proteômica/métodos , Análise de Sequência de DNA , Relação Estrutura-Atividade , Toxinas Biológicas/química , Proteínas Virais/química
5.
Viruses ; 13(2)2021 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-33668618

RESUMO

The Yersinia bacteriophages fPS-2, fPS-65, and fPS-90, isolated from pig stools, have long contractile tails and elongated heads, and they belong to genus Tequatroviruses in the order Caudovirales. The phages exhibited relatively wide host ranges among Yersinia pseudotuberculosis and related species. One-step growth curve experiments revealed that the phages have latent periods of 50-80 min with burst sizes of 44-65 virions per infected cell. The phage genomes consist of circularly permuted dsDNA of 169,060, 167,058, and 167,132 bp in size, respectively, with a G + C content 35.3%. The number of predicted genes range from 267 to 271. The phage genomes are 84-92% identical to each other and ca 85% identical to phage T4. The phage receptors were identified by whole genome sequencing of spontaneous phage-resistant mutants. The phage-resistant strains had mutations in the ompF, galU, hldD, or hldE genes. OmpF is a porin, and the other genes encode lipopolysaccharide (LPS) biosynthetic enzymes. The ompF, galU, and hldE mutants were successfully complemented in trans with respective wild-type genes. The host recognition was assigned to long tail fiber tip protein Gp38, analogous to that of T-even phages such as Salmonella phage S16, specifically to the distal ß-helices connecting loops.


Assuntos
Proteínas de Bactérias/metabolismo , Bacteriófagos/isolamento & purificação , Fezes/virologia , Lipopolissacarídeos/metabolismo , Porinas/metabolismo , Receptores Virais/metabolismo , Yersinia pestis/virologia , Yersinia pseudotuberculosis/virologia , Animais , Proteínas de Bactérias/genética , Bacteriófagos/classificação , Bacteriófagos/genética , Bacteriófagos/fisiologia , Composição de Bases , Genoma Viral , Especificidade de Hospedeiro , Filogenia , Porinas/genética , Receptores Virais/genética , Suínos , Yersinia pestis/genética , Yersinia pestis/metabolismo , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/metabolismo
6.
Front Microbiol ; 11: 1356, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32636826

RESUMO

We report here the complete genome sequence and characterization of Yersinia bacteriophage vB_YenP_ϕ80-18. ϕ80-18 was isolated in 1991 using a Y. enterocolitica serotype O:8 strain 8081 as a host from a sewage sample in Turku, Finland, and based on its morphological and genomic features is classified as a podovirus. The genome is 42 kb in size and has 325 bp direct terminal repeats characteristic for podoviruses. The genome contains 57 predicted genes, all encoded in the forward strand, of which 29 showed no similarity to any known genes. Phage particle proteome analysis identified altogether 24 phage particle-associated proteins (PPAPs) including those identified as structural proteins such as major capsid, scaffolding and tail component proteins. In addition, also the DNA helicase, DNA ligase, DNA polymerase, 5'-exonuclease, and the lytic glycosylase proteins were identified as PPAPs, suggesting that they might be injected together with the phage genome into the host cell to facilitate the take-over of the host metabolism. The phage-encoded RNA-polymerase and DNA-primase were not among the PPAPs. Promoter search predicted the presence of four phage and eleven host RNA polymerase -specific promoters in the genome, suggesting that early transcription of the phage is host RNA-polymerase dependent and that the phage RNA polymerase takes over later. The phage tolerates pH values between 2 and 12, and is stable at 50°C but is inactivated at 60°C. It grows slowly with a 50 min latent period and has apparently a low burst size. Electron microscopy revealed that the phage has a head diameter of about 60 nm, and a short tail of 20 nm. Whole-genome phylogenetic analysis confirmed that ϕ80-18 belongs to the Autographivirinae subfamily of the Podoviridae family, that it is 93.2% identical to Yersinia phage fHe-Yen3-01. Host range analysis showed that ϕ80-18 can infect in addition to Y. enterocolitica serotype O:8 strains also strains of serotypes O:4, O:4,32, O:20 and O:21, the latter ones representing similar to Y. enterocolitica serotype O:8, the American pathogenic biotype 1B strains. In conclusion, the phage ϕ80-18 is a promising candidate for the biocontrol of the American biotype 1B Y. enterocolitica.

7.
Viruses ; 12(6)2020 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-32486497

RESUMO

Acinetobacter baumannii is an opportunistic pathogen that presents a serious clinical challenge due to its increasing resistance to all available antibiotics. Phage therapy has been introduced recently to treat antibiotic-incurable A. baumannii infections. In search for new A. baumannii specific bacteriophages, 20 clinical A. baumannii strains were used in two pools in an attempt to enrich phages from sewage. The enrichment resulted in induction of resident prophage(s) and three temperate bacteriophages, named vB_AbaS_fEg-Aba01, vB_AbaS_fLi-Aba02 and vB_AbaS_fLi-Aba03, all able to infect only one strain (#6597) of the 20 clinical strains, were isolated. Morphological characteristics obtained by transmission electron microscopy together with the genomic information revealed that the phages belong to the family Siphoviridae. The ca. 35 kb genomic sequences of the phages were >99% identical to each other. The linear ds DNA genomes of the phages contained 10 nt cohesive end termini, 52-54 predicted genes, an attP site and one tRNA gene each. A database search revealed an >99% identical prophage in the genome of A. baumannii strain AbPK1 (acc. no. CP024576.1). Over 99% identical prophages were also identified from two of the original 20 clinical strains (#5707 and #5920) and both were shown to be spontaneously inducible, thus very likely being the origins of the isolated phages. The phage vB_AbaS_fEg-Aba01 was also able to lysogenize the susceptible strain #6597 demonstrating that it was fully functional. The phages showed a very narrow host range infecting only two A. baumannii strains. In conclusion, we have isolated and characterized three novel temperate Siphoviridae phages that infect A. baumannii.


Assuntos
Acinetobacter baumannii/virologia , Siphoviridae/fisiologia , DNA Viral/genética , DNA Viral/isolamento & purificação , Genoma Viral/genética , Lisogenia , Microscopia Eletrônica de Transmissão , Filogenia , Análise de Sequência de DNA , Siphoviridae/genética , Siphoviridae/isolamento & purificação , Siphoviridae/patogenicidade , Ensaio de Placa Viral , Ativação Viral
8.
Viruses ; 12(6)2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32517038

RESUMO

YerA41 is a Myoviridae bacteriophage that was originally isolated due its ability to infect Yersinia ruckeri bacteria, the causative agent of enteric redmouth disease of salmonid fish. Several attempts to determine its genomic DNA sequence using traditional and next generation sequencing technologies failed, indicating that the phage genome is modified in such a way that it is an unsuitable template for PCR amplification and for conventional sequencing. To determine the YerA41 genome sequence, we performed RNA-sequencing from phage-infected Y. ruckeri cells at different time points post-infection. The host-genome specific reads were subtracted and de novo assembly was performed on the remaining unaligned reads. This resulted in nine phage-specific scaffolds with a total length of 143 kb that shared only low level and scattered identity to known sequences deposited in DNA databases. Annotation of the sequences revealed 201 predicted genes, most of which found no homologs in the databases. Proteome studies identified altogether 63 phage particle-associated proteins. The RNA-sequencing data were used to characterize the transcriptional control of YerA41 and to investigate its impact on the bacterial gene expression. Overall, our results indicate that RNA-sequencing can be successfully used to obtain the genomic sequence of non-sequencable phages, providing simultaneous information about the phage-host interactions during the process of infection.


Assuntos
Bacteriófagos/genética , Genoma Viral , Yersinia ruckeri/virologia , Bacteriófagos/classificação , Bacteriófagos/isolamento & purificação , Filogenia , RNA Viral/genética , Análise de Sequência de RNA
9.
Viruses ; 12(5)2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32429141

RESUMO

The lytic phage, fHe-Kpn01 was isolated from sewage water using an extended-spectrum beta-lactamase-producing strain of Klebsiella pneumoniae as a host. The genome is 43,329 bp in size and contains direct terminal repeats of 222 bp. The genome contains 56 predicted genes, of which proteomics analysis detected 29 different proteins in purified phage particles. Comparison of fHe-Kpn01 to other phages, both morphologically and genetically, indicated that the phage belongs to the family Podoviridae and genus Drulisvirus. Because fHe-Kpn01 is strictly lytic and does not carry any known resistance or virulence genes, it is suitable for phage therapy. It has, however, a narrow host range since it infected only three of the 72 tested K. pneumoniae strains, two of which were of capsule type KL62. After annotation of the predicted genes based on the similarity to genes of known function and proteomics results on the virion-associated proteins, 22 gene products remained annotated as hypothetical proteins of unknown function (HPUF). These fHe-Kpn01 HPUFs were screened for their toxicity in Escherichia coli. Three of the HPUFs, encoded by the genes g10, g22, and g38, were confirmed to be toxic.


Assuntos
Bacteriófagos/metabolismo , Klebsiella pneumoniae/virologia , Podoviridae/metabolismo , Proteínas Virais/toxicidade , Sequência de Aminoácidos , Antibacterianos/química , Cápsulas Bacterianas/genética , Bacteriófagos/classificação , Bacteriófagos/isolamento & purificação , Bacteriófagos/fisiologia , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Genoma Viral/genética , Especificidade de Hospedeiro , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Filogenia , Podoviridae/classificação , Podoviridae/isolamento & purificação , Podoviridae/fisiologia , Esgotos/virologia , Proteínas Virais/química , Proteínas Virais/genética , Vírion/ultraestrutura , Resistência beta-Lactâmica
10.
Viruses ; 11(11)2019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31739448

RESUMO

The rapid emergence of antibiotic resistance among many pathogenic bacteria has created a profound need to discover new alternatives to antibiotics. Bacteriophages, the viruses of microbes, express special proteins to overtake the metabolism of the bacterial host they infect, the best known of which are involved in bacterial lysis. However, the functions of majority of bacteriophage encoded gene products are not known, i.e., they represent the hypothetical proteins of unknown function (HPUFs). In the current study we present a phage genomics-based screening approach to identify phage HPUFs with antibacterial activity with a long-term goal to use them as leads to find unknown targets to develop novel antibacterial compounds. The screening assay is based on the inhibition of bacterial growth when a toxic gene is expression-cloned into a plasmid vector. It utilizes an optimized plating assay producing a significant difference in the number of transformants after ligation of the toxic and non-toxic genes into a cloning vector. The screening assay was first tested and optimized using several known toxic and non-toxic genes. Then, it was applied to screen 94 HPUFs of bacteriophage φR1-RT, and identified four HPUFs that were toxic to Escherichia coli. This optimized assay is in principle useful in the search for bactericidal proteins of any phage, and also opens new possibilities to understanding the strategies bacteriophages use to overtake bacterial hosts.


Assuntos
Bactérias/virologia , Bacteriólise/genética , Bacteriófagos/fisiologia , Proteômica/métodos , Proteínas Virais/genética , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Escherichia coli/virologia , Modelos Moleculares , Conformação Proteica , Relação Estrutura-Atividade , Proteínas Virais/química
11.
Nucleic Acids Res ; 46(9): 4649-4661, 2018 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-29294068

RESUMO

The phage Mu DNA transposition system provides a versatile species non-specific tool for molecular biology, genetic engineering and genome modification applications. Mu transposition is catalyzed by MuA transposase, with DNA cleavage and integration reactions ultimately attaching the transposon DNA to target DNA. To improve the activity of the Mu DNA transposition machinery, we mutagenized MuA protein and screened for hyperactivity-causing substitutions using an in vivo assay. The individual activity-enhancing substitutions were mapped onto the MuA-DNA complex structure, containing a tetramer of MuA transposase, two Mu end segments and a target DNA. This analysis, combined with the varying effect of the mutations in different assays, implied that the mutations exert their effects in several ways, including optimizing protein-protein and protein-DNA contacts. Based on these insights, we engineered highly hyperactive versions of MuA, by combining several synergistically acting substitutions located in different subdomains of the protein. Purified hyperactive MuA variants are now ready for use as second-generation tools in a variety of Mu-based DNA transposition applications. These variants will also widen the scope of Mu-based gene transfer technologies toward medical applications such as human gene therapy. Moreover, the work provides a platform for further design of custom transposases.


Assuntos
Elementos de DNA Transponíveis , Transposases/genética , Transposases/metabolismo , Substituição de Aminoácidos , Animais , Células Cultivadas , Engenharia Genética , Genoma , Camundongos , Modelos Moleculares , Mutação , Transposases/química , Transposases/isolamento & purificação
12.
Nat Commun ; 8(1): 1915, 2017 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-29203765

RESUMO

Eukaryotic organisms are continuously exposed to bacteriophages, which are efficient gene transfer agents in bacteria. However, bacteriophages are considered not to pass the eukaryotic cell membrane and enter nonphagocytic cells. Here we report the binding and penetration of Escherichia coli PK1A2 bacteriophage into live eukaryotic neuroblastoma cells in vitro. The phage interacts with cell surface polysialic acid, which shares structural similarity with the bacterial phage receptor. Using fluorescence and electron microscopy, we show that phages are internalized via the endolysosomal route and persist inside the human cells up to one day without affecting cell viability. Phage capsid integrity is lost in lysosomes, and the phage DNA is eventually degraded. We did not detect the entry of phage DNA into the nucleus; however, we speculate that this might occur as a rare event, and propose that this potential mechanism could explain prokaryote-eukaryote gene flow.


Assuntos
Bacteriófagos/metabolismo , Endossomos/metabolismo , Escherichia coli/virologia , Células Eucarióticas/metabolismo , Lisossomos/metabolismo , Neuroblastoma/metabolismo , Ácidos Siálicos/metabolismo , Bacteriófagos/ultraestrutura , Capsídeo/metabolismo , Capsídeo/ultraestrutura , Linhagem Celular Tumoral , DNA Viral/metabolismo , Endocitose , Endossomos/ultraestrutura , Células Eucarióticas/ultraestrutura , Fluxo Gênico , Humanos , Lisossomos/ultraestrutura , Microscopia Eletrônica , Microscopia de Fluorescência , Neuroblastoma/ultraestrutura
13.
Mol Microbiol ; 103(6): 1065-1091, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28010054

RESUMO

In bacteria, the RNA chaperone Hfq enables pairing of small regulatory RNAs with their target mRNAs and therefore is a key player of post-transcriptional regulation network. As a global regulator, Hfq is engaged in the adaptation to external environment, regulation of metabolism and bacterial virulence. In this study we used RNA-sequencing and quantitative proteomics (LC-MS/MS) to elucidate the role of this chaperone in the physiology and virulence of Yersinia enterocolitica serotype O:3. This global approach revealed the profound impact of Hfq on gene and protein expression. Furthermore, the role of Hfq in the cell morphology, metabolism, cell wall integrity, resistance to external stresses and pathogenicity was evaluated. Importantly, our results revealed that several alterations typical for the hfq-negative phenotype were due to derepression of the transcriptional factor RovM. The overexpression of RovM caused by the loss of Hfq chaperone resulted in extended growth defect, alterations in the lipid A structure, motility and biofilm formation defects, as well as changes in mannitol utilization. Furthermore, in Y. enterocolitica RovM only in the presence of Hfq affected the abundance of RpoS. Finally, the impact of hfq and rovM mutations on the virulence was assessed in the mouse infection model.


Assuntos
Proteínas de Bactérias/genética , Fator Proteico 1 do Hospedeiro/genética , Chaperonas Moleculares/genética , RNA Longo não Codificante/genética , Pequeno RNA não Traduzido/genética , Sequências Reguladoras de Ácido Ribonucleico/genética , Fator sigma/metabolismo , Fatores de Transcrição/genética , Yersinia enterocolitica/genética , Yersinia enterocolitica/patogenicidade , Animais , Proteínas de Bactérias/metabolismo , Sequência de Bases , Biofilmes/crescimento & desenvolvimento , Parede Celular/genética , Gastroenterite/microbiologia , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/metabolismo , Lipídeo A/metabolismo , Manitol/metabolismo , Espectrometria de Massas , Camundongos , Chaperonas Moleculares/metabolismo , Proteoma/genética , Proteômica , Análise de Sequência de RNA , Fatores de Transcrição/metabolismo , Yersiniose/microbiologia , Yersinia enterocolitica/classificação
14.
Methods Mol Biol ; 1498: 309-320, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27709584

RESUMO

Archaea constitute the third domain of life, but studies on their physiology and other features have lagged behind bacteria and eukarya, largely due to the challenging biology of archaea and concomitant difficulties in methods development. The use of genome-wide en masse insertion mutagenesis is one of the most efficient means to discover the genes behind various biological functions, and such a methodology is described in this chapter for a model archaeon Haloferax volcanii. The strategy successfully employs efficient in vitro transposition in combination with gene targeting in vivo via homologous recombination. The methodology is general and should be transferable to other archaeal species.


Assuntos
Archaea/genética , Elementos de DNA Transponíveis/genética , Genes Arqueais/genética , Mutagênese Insercional/genética , Proteínas Arqueais/genética , Eucariotos/genética , Haloferax volcanii/genética , Recombinação Homóloga/genética
15.
Stem Cell Res Ther ; 7(1): 113, 2016 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-27528376

RESUMO

BACKGROUND: In order to develop novel clinical applications and to gain insights into possible therapeutic mechanisms, detailed molecular characterization of human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) is needed. Neural cell adhesion molecule (NCAM, CD56) is a transmembrane glycoprotein modulating cell-cell and cell-matrix interactions. An additional post-translational modification of NCAM is the α2,8-linked polysialic acid (polySia). Because of its background, NCAM is often considered a marker of neural lineage commitment. Generally, hBM-MSCs are considered to be devoid of NCAM expression, but more rigorous characterization is needed. METHODS: We have studied NCAM and polySia expression in five hBM-MSC lines at mRNA and protein levels. Cell surface localization was confirmed by immunofluorescence staining and expression frequency in the donor-specific lines by flow cytometry. For the detection of poorly immunogenic polySia, a fluorochrome-tagged catalytically defective enzyme was employed. RESULTS: All five known NCAM isoforms are expressed in these cells at mRNA level and the three main isoforms are present at protein level. Both polysialyltransferases, generally responsible for NCAM polysialylation, are expressed at mRNA level, but only very few cells express polySia at the cell surface. CONCLUSIONS: Our results underline the need for a careful control of methods and conditions in the characterization of MSCs. This study shows that, against the generally held view, clinical-grade hBM-MSCs do express NCAM. In contrast, although both polysialyltransferase genes are transcribed in these cells, very few express polySia at the cell surface. NCAM and polySia represent new candidate molecules for influencing MSC interactions.


Assuntos
Medula Óssea/metabolismo , Antígeno CD56/metabolismo , Células-Tronco Mesenquimais/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Ácidos Siálicos/metabolismo , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Linhagem da Célula/fisiologia , Humanos , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Sialiltransferases/metabolismo
16.
BMC Biol ; 12: 103, 2014 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-25488358

RESUMO

BACKGROUND: Archaea share fundamental properties with bacteria and eukaryotes. Yet, they also possess unique attributes, which largely remain poorly characterized. Haloferax volcanii is an aerobic, moderately halophilic archaeon that can be grown in defined media. It serves as an excellent archaeal model organism to study the molecular mechanisms of biological processes and cellular responses to changes in the environment. Studies on haloarchaea have been impeded by the lack of efficient genetic screens that would facilitate the identification of protein functions and respective metabolic pathways. RESULTS: Here, we devised an insertion mutagenesis strategy that combined Mu in vitro DNA transposition and homologous-recombination-based gene targeting in H. volcanii. We generated an insertion mutant library, in which the clones contained a single genomic insertion. From the library, we isolated pigmentation-defective and auxotrophic mutants, and the respective insertions pinpointed a number of genes previously known to be involved in carotenoid and amino acid biosynthesis pathways, thus validating the performance of the methodologies used. We also identified mutants that had a transposon insertion in a gene encoding a protein of unknown or putative function, demonstrating that novel roles for non-annotated genes could be assigned. CONCLUSIONS: We have generated, for the first time, a random genomic insertion mutant library for a halophilic archaeon and used it for efficient gene discovery. The library will facilitate the identification of non-essential genes behind any specific biochemical pathway. It represents a significant step towards achieving a more complete understanding of the unique characteristics of halophilic archaea.


Assuntos
Proteínas Arqueais/genética , Elementos de DNA Transponíveis/genética , Biblioteca Gênica , Haloferax volcanii/genética , Mutagênese Insercional , Carotenoides/biossíntese , Clonagem Molecular , Marcação de Genes , Estudos de Associação Genética , Redes e Vias Metabólicas , Plasmídeos/genética , Recombinação Genética
17.
Mob DNA ; 1(1): 24, 2010 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-21110848

RESUMO

BACKGROUND: Completed genome projects have revealed an astonishing diversity of transposable genetic elements, implying the existence of novel element families yet to be discovered from diverse life forms. Concurrently, several better understood transposon systems have been exploited as efficient tools in molecular biology and genomics applications. Characterization of new mobile elements and improvement of the existing transposition technology platforms warrant easy-to-use assays for the quantitative analysis of DNA transposition. RESULTS: Here we developed a universal in vivo platform for the analysis of transposition frequency with class II mobile elements, i.e., DNA transposons. For each particular transposon system, cloning of the transposon ends and the cognate transposase gene, in three consecutive steps, generates a multifunctional plasmid, which drives inducible expression of the transposase gene and includes a mobilisable lacZ-containing reporter transposon. The assay scores transposition events as blue microcolonies, papillae, growing within otherwise whitish Escherichia coli colonies on indicator plates. We developed the assay using phage Mu transposition as a test model and validated the platform using various MuA transposase mutants. For further validation and to illustrate universality, we introduced IS903 transposition system components into the assay. The developed assay is adjustable to a desired level of initial transposition via the control of a plasmid-borne E. coli arabinose promoter. In practice, the transposition frequency is modulated by varying the concentration of arabinose or glucose in the growth medium. We show that variable levels of transpositional activity can be analysed, thus enabling straightforward screens for hyper- or hypoactive transposase mutants, regardless of the original wild-type activity level. CONCLUSIONS: The established universal papillation assay platform should be widely applicable to a variety of mobile elements. It can be used for mechanistic studies to dissect transposition and provides a means to screen or scrutinise transposase mutants and genes encoding host factors. In succession, improved versions of transposition systems should yield better tools for molecular biology and offer versatile genome modification vehicles for many types of studies, including gene therapy and stem cell research.

18.
Anal Biochem ; 388(1): 71-80, 2009 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-19454214

RESUMO

Random mutagenesis methods constitute a valuable protein modification toolbox with applications ranging from protein engineering to directed protein evolution studies. Although a variety of techniques are currently available, the field is lacking studies that would directly compare the performance parameters and operational range of different methods. In this study, we have scrutinized several of the most commonly used random mutagenesis techniques by critically evaluating popular error-prone polymerase chain reaction (PCR) protocols as well as hydroxylamine and a mutator Escherichia coli strain mutagenesis methods. Relative mutation frequencies were analyzed using a reporter plasmid that allowed direct comparison of the methods. Error-prone PCR methods yielded the highest mutation rates and the widest operational ranges, whereas the chemical and biological methods generated a low level of mutations and exhibited a narrow range of operation. The repertoire of transitions versus transversions varied among the methods, suggesting the use of a combination of methods for high-diversity full-scale mutagenesis. Using the parameters defined in this study, the evaluated mutagenesis methods can be used for controlled mutagenesis, where the intended average frequency of induced mutations can be adjusted to a desirable level.


Assuntos
Escherichia coli/enzimologia , Hidroxilamina/química , Mutagênese , Reação em Cadeia da Polimerase/métodos , Substituição de Aminoácidos , DNA Bacteriano , Frequência do Gene , Plasmídeos , Engenharia de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Taq Polimerase/genética , Taq Polimerase/metabolismo
19.
Nucleic Acids Res ; 36(22): e148, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18953026

RESUMO

Genomic parasites have evolved distinctive lifestyles to optimize replication in the context of the genomes they inhabit. Here, we introduced new DNA into eukaryotic cells using bacteriophage Mu DNA transposition complexes, termed 'transpososomes'. Following electroporation of transpososomes and selection for marker gene expression, efficient integration was verified in yeast, mouse and human genomes. Although Mu has evolved in prokaryotes, strong biases were seen in the target site distributions in eukaryotic genomes, and these biases differed between yeast and mammals. In Saccharomyces cerevisiae transposons accumulated outside of genes, consistent with selection against gene disruption. In mouse and human cells, transposons accumulated within genes, which previous work suggests is a favorable location for efficient expression of selectable markers. Naturally occurring transposons and viruses in yeast and mammals show related, but more extreme, targeting biases, suggesting that they are responding to the same pressures. These data help clarify the constraints exerted by genome structure on genomic parasites, and illustrate the wide utility of the Mu transpososome technology for gene transfer in eukaryotic cells.


Assuntos
Bacteriófago mu/genética , Elementos de DNA Transponíveis , Técnicas de Transferência de Genes , Animais , Linhagem Celular , Mapeamento Cromossômico , Eletroporação , Células-Tronco Embrionárias/metabolismo , Marcadores Genéticos , Genoma Fúngico , Genoma Humano , Genômica , Células HeLa , Humanos , Camundongos , Saccharomyces cerevisiae/genética
20.
Microbiology (Reading) ; 151(Pt 4): 1209-18, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15817788

RESUMO

Transposon mutagenesis is a powerful technique for generating collections of insertion mutants for genetic studies. This paper describes how phage Mu DNA transposition complexes, transpososomes, can be exploited for gene delivery to efficiently introduce selectable markers to genomes of Gram-positive bacteria. Mu transpososomes were assembled in vitro with custom-designed mini-Mu transposons, concentrated, and electroporated into cells of three Gram-positive bacterial species: Staphylococcus aureus, Streptococcus pyogenes and Streptococcus suis. Within cells, the complexes reproduced an authentic DNA transposition reaction and integrated the delivered transposons into the bacterial genomes, yielding single-copy insertions. The integration efficiency among different species and strains of Gram-positive bacteria ranged from 1x10(1) to 2x10(4) c.f.u. (mug introduced transposon DNA)(-1). The strategy should be applicable to a variety of other Gram-positive species after initial optimization of certain key factors affecting transposon delivery, such as the preparation method of competent cells and physical parameters of electroporation. This study extends the scope of the Mu transpososome delivery-based genomic DNA integration strategy to Gram-positive bacteria. Thus, a straightforward generation of sizeable mutant banks is feasible for these bacteria, potentiating several types of genomic-level approaches for studies of a variety of important bacterial processes, such as pathogenicity.


Assuntos
Bacteriófago mu/genética , Elementos de DNA Transponíveis/genética , Biblioteca Gênica , Bactérias Gram-Positivas/genética , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Bacterianos/genética , DNA Bacteriano/genética , Eletroporação , Técnicas Genéticas , Genoma Bacteriano , Mutagênese Insercional , Staphylococcus aureus/genética , Streptococcus pyogenes/genética , Streptococcus suis/genética , Integração Viral
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